Abnormal sexual development related gene capture kit and its application
A gene capture and kit technology, which can be used in biochemical equipment and methods, DNA/RNA fragments, and microbial determination/inspection. , high accuracy, and the effect of improving population quality
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Embodiment 1
[0098] Example 1. Preparation of a complete set of reagents and kits for detecting sex chromosome abnormality-related genes
[0099] 1. Design and preparation of sex chromosome abnormality-related gene capture probes
[0100] 1. Design and synthesize 277 probes based on the whole exon sequences of 277 sex chromosome abnormality-related genes (design 120bp probe sequences for non-repeated regions in each region, and move each sequence along the position of the gene) .
[0101] Each of the 277 probes is composed of DNA fragment 1, probe fragment and DNA fragment 2 (each probe is DNA fragment 1, probe fragment and DNA fragment 2 in turn from the 5' end to the 3' end, 277 DNA Fragment 1 among the probes is the same, and DNA Fragment 2 is the same among the 277 probes). The nucleotide sequence of DNA fragment 1 is 5'-GACTACATGGGACAT-3'. The nucleotide sequence of DNA fragment 2 is 5'-GGAACCTACGACGTA-3'. The probe fragments in the 277 probes are shown in Table 1.
[0102] Table...
Embodiment 2
[0129] Example 2. Establishment of the method of using the kit for capturing genes related to abnormal sexual development
[0130] 1. Sample library preparation
[0131] 1. With the informed consent of the patient, extract the genomic DNA from the peripheral blood of the patient to be tested.
[0132] 2. Fragment the genomic DNA obtained in step 1 to 200-400bp fragments, and use the NGS Fast DNALibrary Prep Set for Illumina kit (Kangwei reagent, catalog number: CW2585M) to construct a library to obtain a DNA gene library.
[0133] 2. Sample enrichment hybridization
[0134] 1. Preparation of hybridization solution
[0135] Hybridization solution (100μl): 250ng DNA library prepared in step 1, 10μl enrichment buffer BL, 5μl biotin-labeled probe set obtained in step 1 (probe concentration is 100ng / μL), 37μL preheated enrichment Buffer solution HY (preheated at 65°C, shake well to resuspend the pellet before use).
[0136] 2. Hybridization
[0137] Take the hybridization solu...
Embodiment 3
[0168] Example 3, Sequencing Effect Verification
[0169] With the informed consent of the patients, peripheral blood samples from 2 patients with genes related to chromosomal abnormalities were collected and tested according to the method in Example 2.
[0170] The results are shown in Table 2. The results confirmed that more than 99% of the original short sequences can be compared back to the reference sequence of the target region, the capture efficiency is above 35%, and the average sequencing depth of the target region is greater than 250X, fully meeting the requirements of general genetic disease diagnosis.
[0171] Table 2 Data Analysis Results
[0172]
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